The invention relates to methods and compositions for screening for compounds that inhibit or modulate phosphorylation of Wee1 fusion proteins by kinase Cds1.
Cell cycle checkpoints ensure that chromosomal DNA is replicated and repaired prior to nuclear division (Hartwell et al, Science, 246:629, 1989; L. Hartwell, Cell, 71:543, 1992; and S. J. Elledge, Science, 274:1664, 1996). In mammalian cells, loss of checkpoint control results in rearrangements, amplification, and loss of chromosomes, events that are causally associated with cancer. In yeasts, checkpoint controls are vital for survival when DNA is damaged or replication is inhibited.
Cdc2, the cyclin-dependent kinase that initiates mitosis, is the ultimate target of the DNA replication and repair checkpoints and is regulated by checkpoint kinases that couple mitosis to the completion of DNA replication and repair. The repair checkpoint kinase Chk1 regulates Cdc25, a phosphatase which activates Cdc2. In the fission yeast Schizosaccharomyces pombe, Cdc2 is inhibited by phosphorylation on tyrosine-15. This phosphorylation is catalyzed by the kinases Wee1 and Mik1 and reversed by the phosphatase Cdc25. Inhibitory phosphorylation of Cdc2 is crucial for replication and repair checkpoints in fission yeast and human cells (Enoch et al, Cell, 60:665, 1990; Lundgren et al, Cell, 64:1111, 1991; Jin et al, J. Cell Biol., 134:963 (1996); Blasina et al, Mol. Biol. Cell, 8:1013, 1997; and Rhind et al, Genes Dev., 11:504, 1997). Chk1 apparently enforces the DNA repair checkpoint by phosphorylating and inhibiting Cdc25 (Rhind et al, Genes Dev., 11:504, 1997; Furnari et al, Science, 277:1495, 1997; Sanchez et al, Science, 277:1497 (1997); and Peng et al, Science, 277:1501, 1997). Chk1 is only required for the repair checkpoint, not the replication checkpoint evoked by hydroxyurea (HU)(Walworth et al, Nature, 363:368, 1993; and al-Khodairy et al, Mol, Biol. Cell, 5:147, 1994).
Thus, the kinase Wee1 participates in the regulation of cell cycle checkpoints during mitosis by catalyzing phosphorylation of the cyclin-dependent kinase that initiates mitosis, Cdc2. The role of Wee1 in these regulatory processes for controlling cell cycle are not well characterized.
The kinase Cds1 has also been implicated in the checkpoint process, although its role is not understood (Marukami et al, Nature, 374: 817, 1995).
There is a need for systems to characterize the role of Wee1 and Cds1 in regulation of cell cycle, and for screening for compounds which inhibit or modulate the effectors of these processes, particularly inhibitors of Wee1 or Cds1 function.
It has now been discovered that Wee1 is phosphorylated by Cds1, and that Cds1 has an important role in the regulation of the replication checkpoint for mitosis. It has also been discovered that Wee1 can be used in various forms, including truncated proteins based on Wee1, in compositions and methods for evaluating the components of the Wee1-dependent processes related to replication checkpoint, particularly Cds1.
The invention therefore describes various nucleic acids which encode a truncated Wee1 protein, including expression vectors containing the nucleic acids for expressing the Wee1 protein. Also described are various forms of truncated Wee1 protein, particularly fusion proteins containing a Wee1 amino acid residue sequence which defines the functional site for phosphorylation by Cds1.
The invention describes methods for identifying compounds which modulate Wee1 function, or which modulate the ability of Cds1 to phosphorylate Wee1.
Other uses will be apparent to one skilled in the art in light of the present disclosures.